Genetic examinations by analysis of nucleic acids are widely employed for medical, research, and industrial applications with recent progress in technologies of genetic manipulation, genetic recombination, and the like. These examinations involve the detection and quantification of the presence of a target nucleic acid having a target nucleotide sequence in a sample, and are applied in various fields, not only in the diagnoses and treatment of diseases, but also in examination of food. For example, genetic examinations for detecting congenital or acquired mutant genes, virus-related genes, and others are carried out for diagnosis of diseases, such as genetic diseases, tumors, and infections. Analysis of genetic polymorphisms, including single nucleotide polymorphism (SNP), is also applied not only to clinical examinations and academic research, but also to quality checks and traceability of foods and others.
Samples which are subject to gene analysis are often available only in trace amounts, like specimens in forensic or clinical examinations. For this reason, genome fragments containing a target nucleic acid are usually amplified in advance and the amplified genome fragments are employed to detect and quantify the target nucleic acid. Often, the amplification of the target nucleic acid is performed by means of the Polymerase Chain Reaction (PCR).
By means of PCR it is possible to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified.
PCR is often used in the form of real-time PCR, where amplification and detection are closely coupled. Several devices for real-time PCR are commercially available, such as “Roche Light Cycler”, “Cepheid Smart Cycler”, and the like. An alternative to real-time PCR is standard or endpoint PCR where the detection step follows after the completion of the PCR. When using standard or endpoint PCR, detection of amplified DNA is generally performed by gel electrophoresis, capillary electrophoresis, capillary gel electrophoresis or hybridization on dot blots or microarrays.
For a number of diagnostic applications, sensitive and simultaneous measurements of the presence of a number of different specific DNA target sequences are required. Although real-time PCR meets these requirements for a few specific parameters, it does not allow the measurement of a large number of analytes simultaneously within the same reaction due to the limited amount of different available fluorescent dyes and technical difficulties with detectors for more than four different fluorescent dyes. Currently available instruments allow the simultaneous detection of at most four different DNA target sequences within one reaction when using real-time PCR. The combination of a standard or endpoint PCR with a subsequent hybridization reaction does allow the simultaneous analysis of a larger number of analytes, but requires handling of the amplified DNA target sequences within the liquid sample which greatly increases the risk of sample cross contamination.
Thus, the object of the present invention is to provide a reaction vessel for a PCR device, a PCR device including such a reaction vessel and a method for performing PCR including detection of the amplified PCR products that overcome the above described drawbacks of conventional PCR devices and methods.